Dysfunction of cerebellar microglia in Ataxia-telangiectasia.

Ataxia-telangiectasia (A-T) is a multisystem autosomal recessive disease caused by mutations in the ATM gene and characterized by cerebellar atrophy, progressive ataxia, immunodeficiency, male and female sterility, radiosensitivity, cancer predisposition, growth retardation, insulin-resistant diabetes, and premature aging. ATM phosphorylates more than 1500 target proteins, which are involved in cell cycle control, DNA repair, apoptosis, modulation of chromatin structure, and other cytoplasmic as well as mitochondrial processes. In our quest to better understand the mechanisms by which ATM deficiency causes cerebellar degeneration, we hypothesized that specific vulnerabilities of cerebellar microglia underlie the etiology of A-T. Our hypothesis is based on the recent finding that dysfunction of glial cells affect a variety of process leading to impaired neuronal functionality (Song et al., 2019). Whereas astrocytes and neurons descend from the neural tube, microglia originate from the hematopoietic system, invade the brain at early embryonic stage, and become the innate immune cells of the central nervous system and important participants in development of synaptic plasticity. Here we demonstrate that microglia derived from Atm-/- mouse cerebellum display accelerated cell migration and are severely impaired in phagocytosis, secretion of neurotrophic factors, and mitochondrial activity, suggestive of apoptotic processes. Interestingly, no microglial impairment was detected in Atm-deficient cerebral cortex, and Atm deficiency had less impact on astroglia than microglia. Collectively, our findings validate the roles of glial cells in cerebellar attrition in A-T.

Toward neuroprosthetic real-time communication from in silico to biological neuronal network via patterned optogenetic stimulation.

Restoration of the communication between brain circuitry is a crucial step in the recovery of brain damage induced by traumatic injuries or neurological insults. In this work we present a study of real-time unidirectional communication between a spiking neuronal network (SNN) implemented on digital platform and an in-vitro biological neuronal network (BNN), generating similar spontaneous patterns of activity both spatial and temporal. The communication between the networks was established using patterned optogenetic stimulation via a modified digital light projector (DLP) receiving real-time input dictated by the spiking neurons' state. Each stimulation consisted of a binary image composed of 8 × 8 squares, representing the state of 64 excitatory neurons. The spontaneous and evoked activity of the biological neuronal network was recorded using a multi-electrode array in conjunction with calcium imaging. The image was projected in a sub-portion of the cultured network covered by a subset of the all electrodes. The unidirectional information transmission (SNN to BNN) is estimated using the similarity matrix of the input stimuli and output firing. Information transmission was studied in relation to the distribution of stimulus frequency and stimulus intensity, both regulated by the spontaneous dynamics of the SNN, and to the entrainment of the biological networks. We demonstrate that high information transfer from SNN to BNN is possible and identify a set of conditions under which such transfer can occur, namely when the spiking network synchronizations drive the biological synchronizations (entrainment) and in a linear regime response to the stimuli. This research provides further evidence of possible application of miniaturized SNN in future neuro-prosthetic devices for local replacement of injured micro-circuitries capable to communicate within larger brain networks.

Calcium imaging, MEA recordings, and immunostaining images dataset of neuron-astrocyte networks in culture under the effect of norepinephrine.

Background: Monitoring the activity and morphology of neuron-astrocyte networks in culture is a powerful tool for studying dynamics, structure, and communication in neuron-astrocyte networks independently or as a model of the sub-brain network. These cultures are known to produce stereotypical patterns of activity, e.g., highly synchronized network bursts resembling sleep or seizure states, thus it enables the exploration of behaviors that can relate to brain function and disease. High-resolution microscopy of calcium imaging combined with simultaneous electrical recording provides a comprehensive overview on the network's dynamics. This setup makes it possible to apply global perturbations of electrical and chemical stimulation on the cultures during the recording task and to record the effects on network activity on-line. Morphological changes in the cultures can be obtained to have a complete dataset for structure-function study of neuron-astrocyte networks in vitro. Findings: The 4 TB of data presented here was recorded and imaged as part of an accompanying study looking at in vitro structure-function of neuron-astrocyte networks. Simultaneous optical (calcium imaging) and electrical (micro-electrode array) recordings lasted 5-12 minutes and included spontaneous activity recording, electrical and chemical stimulation of neuron-astrocyte, and isolated astrocyte cultures. The data include activity recordings of 58 different cultures, with 1-2 regions of interest recorded for each culture. Production procedures, experimental protocols, and reuse options are included. The data have been suitable to reveal changes in the activity and morphology of the cultures and enabled observation and analysis of neuron-astrocyte and isolated astrocyte culture behaviors under the applied perturbations. Conclusions: Our dataset is sufficient to show significant changes in activity and morphology of neuron-astrocyte networks in culture under the applied stimulations. More than 100 recordings of 58 different cultures give insight of the observation's significance and led to conclusions about astrocyte activity and neuron-astrocyte network communication. Making it available here will allow others to test new tools for calcium imaging analysis and extracellular neuronal voltage recordings.

Activity changes in neuron-astrocyte networks in culture under the effect of norepinephrine.

The concerted activity of neuron-glia networks is responsible for the fascinating dynamics of brain functions. Although these networks have been extensively investigated using a variety of experimental (in vivo and in vitro) and theoretical models, the manner by which neuron-glia networks interact is not fully understood. In particular, how neuromodulators influence network-level signaling between neurons and astrocytes was poorly addressed. In this work, we investigated global effects of the neuromodulator norepinephrine (NE) on neuron-astrocyte network communication in co-cultures of neurons and astrocytes and in isolated astrocyte networks. Electrical stimulation was used to activate the neuron-astrocyte glutamate-mediated pathway. Our results showed dramatic changes in network activity under applied global perturbations. Under neuromodulation, there was a marked rise in calcium signaling in astrocytes, neuronal spontaneous activity was reduced, and the communication between neuron-astrocyte networks was perturbed. Moreover, in the presence of NE, we observed two astrocyte behaviors based on their coupling to neurons. There were also morphological changes in astrocytes upon application of NE, suggesting a physical cause underlies the change in signaling. Our results shed light on the role of NE in controlling sleep-wake cycles.

Astrocytes restore connectivity and synchronization in dysfunctional cerebellar networks.

Evidence suggests that astrocytes play key roles in structural and functional organization of neuronal circuits. To understand how astrocytes influence the physiopathology of cerebellar circuits, we cultured cells from cerebella of mice that lack the ATM gene. Mutations in ATM are causative of the human cerebellar degenerative disease ataxia-telangiectasia. Cerebellar cultures grown from Atm-/- mice had disrupted network synchronization, atrophied astrocytic arborizations, reduced autophagy levels, and higher numbers of synapses per neuron than wild-type cultures. Chimeric circuitries composed of wild-type astrocytes and Atm-/- neurons were indistinguishable from wild-type cultures. Adult cerebellar characterizations confirmed disrupted astrocyte morphology, increased GABAergic synaptic markers, and reduced autophagy in Atm-/- compared with wild-type mice. These results indicate that astrocytes can impact neuronal circuits at levels ranging from synaptic expression to global dynamics.

Astrocytes from old Alzheimer's disease mice are impaired in Aß uptake and in neuroprotection.

In Alzheimer's disease (AD), astrocytes undergo morphological changes ranging from atrophy to hypertrophy, but the effect of such changes at the functional level is still largely unknown. Here, we aimed to investigate whether alterations in astrocyte activity in AD are transient and depend on their microenvironment, or whether they are irreversible. We established and characterized a new protocol for the isolation of adult astrocytes and discovered that astrocytes isolated from old 5xFAD mice have higher GFAP expression than astrocytes derived from WT mice, as observed in vivo. We found high C1q levels in brain sections from old 5xFAD mice in close vicinity to amyloid plaques and astrocyte processes. Interestingly, while old 5xFAD astrocytes are impaired in uptake of soluble Aß42, this effect was reversed upon an addition of exogenous C1q, suggesting a potential role for C1q in astrocyte-mediated Aß clearance. Our results suggest that scavenger receptor B1 plays a role in C1q-facilitated Aß uptake by astrocytes and that expression of scavenger receptor B1 is reduced in adult old 5xFAD astrocytes. Furthermore, old 5xFAD astrocytes show impairment in support of neuronal growth in co-culture and neurotoxicity concomitant with an elevation in IL-6 expression. Further understanding of the impact of astrocyte impairment on AD pathology may provide insights into the etiology of AD.

Connecting Malfunctioning Glial Cells and Brain Degenerative Disorders.

The DNA damage response (DDR) is a complex biological system activated by different types of DNA damage. Mutations in certain components of the DDR machinery can lead to genomic instability disorders that culminate in tissue degeneration, premature aging, and various types of cancers. Intriguingly, malfunctioning DDR plays a role in the etiology of late onset brain degenerative disorders such as Parkinson's, Alzheimer's, and Huntington's diseases. For many years, brain degenerative disorders were thought to result from aberrant neural death. Here we discuss the evidence that supports our novel hypothesis that brain degenerative diseases involve dysfunction of glial cells (astrocytes, microglia, and oligodendrocytes). Impairment in the functionality of glial cells results in pathological neuro-glial interactions that, in turn, generate a "hostile" environment that impairs the functionality of neuronal cells. These events can lead to systematic neural demise on a scale that appears to be proportional to the severity of the neurological deficit.

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits.

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity. A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

Rapamycin increases neuronal survival, reduces inflammation and astrocyte proliferation after spinal cord injury.

Spinal cord injury (SCI) frequently leads to a permanent functional impairment as a result of the initial injury followed by secondary injury mechanism, which is characterised by increased inflammation, glial scarring and neuronal cell death. Finding drugs that may reduce inflammatory cell invasion and activation to reduce glial scarring and increase neuronal survival is of major importance for improving the outcome after SCI. In the present study, we examined the effect of rapamycin, an mTORC1 inhibitor and an inducer of autophagy, on recovery from spinal cord injury. Autophagy, a process that facilitates the degradation of cytoplasmic proteins, is also important for maintenance of neuronal homeostasis and plays a major role in neurodegeneration after neurotrauma. We examined rapamycin effects on the inflammatory response, glial scar formation, neuronal survival and regeneration in vivo using spinal cord hemisection model in mice, and in vitro using primary cortical neurons and human astrocytes. We show that a single injection of rapamycin, inhibited p62/SQSTM1, a marker of autophagy, inhibited mTORC1 downstream effector p70S6K, reduced macrophage/neutrophil infiltration into the lesion site, microglia activation and secretion of TNFα. Rapamycin inhibited astrocyte proliferation and reduced the number of GFAP expressing cells at the lesion site. Finally, it increased neuronal survival and axonogenesis towards the lesion site. Our study shows that rapamycin treatment increased significantly p-Akt levels at the lesion site following SCI. Similarly, rapamycin treatment of neurons and astrocytes induced p-Akt elevation under stress conditions. Together, these findings indicate that rapamycin is a promising candidate for treatment of acute SCI condition and may be a useful therapeutic agent.

In vitro large-scale experimental and theoretical studies for the realization of bi-directional brain-prostheses.

Brain-machine interfaces (BMI) were born to control "actions from thoughts" in order to recover motor capability of patients with impaired functional connectivity between the central and peripheral nervous system. The final goal of our studies is the development of a new proof-of-concept BMI-a neuromorphic chip for brain repair-to reproduce the functional organization of a damaged part of the central nervous system. To reach this ambitious goal, we implemented a multidisciplinary "bottom-up" approach in which in vitro networks are the paradigm for the development of an in silico model to be incorporated into a neuromorphic device. In this paper we present the overall strategy and focus on the different building blocks of our studies: (i) the experimental characterization and modeling of "finite size networks" which represent the smallest and most general self-organized circuits capable of generating spontaneous collective dynamics; (ii) the induction of lesions in neuronal networks and the whole brain preparation with special attention on the impact on the functional organization of the circuits; (iii) the first production of a neuromorphic chip able to implement a real-time model of neuronal networks. A dynamical characterization of the finite size circuits with single cell resolution is provided. A neural network model based on Izhikevich neurons was able to replicate the experimental observations. Changes in the dynamics of the neuronal circuits induced by optical and ischemic lesions are presented respectively for in vitro neuronal networks and for a whole brain preparation. Finally the implementation of a neuromorphic chip reproducing the network dynamics in quasi-real time (10 ns precision) is presented.

The role of the neuro-astro-vascular unit in the etiology of ataxia telangiectasia.

The growing recognition that brain pathologies do not affect neurons only but rather are, to a large extent, pathologies of glial cells as well as of the vasculature opens to new perspectives in our understanding of genetic disorders of the CNS. To validate the role of the neuron-glial-vascular unit in the etiology of genome instability disorders, we report about cell death and morphological aspects of neuroglia networks and the associated vasculature in a mouse model of Ataxia Telangiectasia (A-T), a human genetic disorder that induces severe motor impairment. We found that A-T-mutated protein deficiency was consistent with aberrant astrocytic morphology and alterations of the vasculature, often accompanied by reactive gliosis. Interestingly similar findings could also be reported in the case of other genetic disorders. These observations bolster the notion that astrocyte-specific pathologies, hampered vascularization and astrocyte-endothelium interactions in the CNS could play a crucial role in the etiology of genome instability brain disorders and could underlie neurodegeneration.